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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/32—Amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/70—Undefined extracts
  • C12N2500/74—Undefined extracts from fungi, e.g. yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/70—Undefined extracts
  • C12N2500/80—Undefined extracts from animals
  • C12N2500/84—Undefined extracts from animals from mammals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
  • C12N2500/92—Medium free of human- or animal-derived components
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/90—Polysaccharides

Definitions

• the present invention relates to the field of media for the culture of cells. More particularly the present invention relates to media for the culture of insect cells.
• Serum in cell culture media, however, has several disadvantages. Serum is comparatively expensive. Since serum is not a defined component, different lots of serum may vary in the concentration of compounds present and thus result in unpredictable culture growth. Serum may also be contaminated with viruses or mycoplasmas. The protein in serum may complicate the purification of cell products from the culture media.
• SUBSTITUTESHEET is able to support primary antibody responses by cultured murine lymphocytes.
• This medium is based on a basal medium supplemented with ⁇ -cyclodextrin, insulin, transferrin, albumin, low density lipoprotein, putrescine and alanine.
• a serum-free medium for large-scale culture of insect (Spodoptera frugiperda. cells was reported in Maiorella et al., (1988) Biotechnology £: 1406-1410.
• the medium contained yeast extract, cod liver oil polyunsaturated fatty acid methyl esters, cholesterol and Tween.
• Murhammer and Goochee (1988) Biotechnology £.: 1411-1418 discloses a medium for the culture of Spodoptera frugiperda that contains serum.
• the present invention provides serum-free media for the culture of insect cells.
• the invention is more particularly pointed out in the appended claims and is described in its preferred embodiments in the following description.
• the serum-free media of the invention are useful for the culture of insect ce * s.
• the media of the invention have b a n found to joe useful in the culture of Drosophila cells, and should have utility in the culture of a wide variety of insect cells.
• Cells may be grown in batch and continuous culture with the serum-free media of the invention. Drosophila cells grown in the media of the invention reach higher cell density and US91/06851
• the media of the invention have a low protein content which is advantageous for culturing cells for the production of recombinant proteins.
• the low protein content of the media reduces the amount of unwanted proteins that have to be separated from the protein product during purification of the protein product. Additionally, the media may be made at a much lower cost than media containing serum.
• the cell culture media are prepared by adding supplements to a basal medium designed for insect cell culture.
• the media are prepared accordance with standard procedures for preparing cell culture media.
• basal medium for use in the serum-free media of the invention is described in Example 1.
• suitable basal media include Grace's (Gibco, Grand Island, new York) and Schneider's (Gibco).
• Yeast hydrolysate is added in the amount of from about 1 to about 10 grams per liter.
• Dextran or albumin are added in the amount of from about 0.1 to about 5 grams per liter.
• Albumin for use in the media is preferably bovine serum albumin, however, albumin from other species is also suitable.
• Dextran for use in the media is preferably dextran having a molecular weight of about 500,000 such as dextran T-500.
• lactalbu in hydrolysate is added in the amount of from about 0.2 to about 5 grams per liter, preferably in the amount of about 5 grams per liter.
• the pH range of the media is preferably in the range of from about 6.3 to about 6.7.
• the osmolarity is preferably in the range of from about 320 to about 360 milliosmoles.
• the basal medium may be stored as a powder at 4°C for one year.
• the complete medium (basal medium with
• added supplements in a liquid form may be stored at 4°C for six months.
• the components in the basal medium are mixed and ball-mill ground to formulate a homogeneous powder.
• the powdered medium is then dispensed into 100L packets and stored at 4°C for up to one year.
• SUBSTITUTESHEET above is added.
• the pH of the medium is adjusted to 6.6 using 10 N NaOH.
• the volume of the medium is brought to 100L by the addition of water.
• the medium may then be sterilized by membrane filtration using a 0.2 micron cellulose acetate filter.
• Medium MR-D1 contains the basal medium of Example 1 supplemented with 5 grams per liter TC lactalbumin hydrolysate (Difco, Detroit, Michigan) , 1 gram per liter TC yeastolate (Difco, Detroit, Michigan) and 1 gram per liter bovine serum albumin (Armour, Kankakee, Illinois) .
• the medium is prepared as follows: For a final volume of 100 liters -
• Medium MR-D2 contains the basal medium of Example 1 supplemented with 5 grams per liter TC yeastolate (Difco, Detroit, Michigan) and 1 gram per liter bovine serum albumin (Armour, Kankakee, Illinois) .
• the medium is prepared as follows:
• Medium MR-D3 contains the basal medium of Example 1 supplemented with 5 grams per liter TC yeastolate (Difco, Detroit, Michigan) and 1 gram per liter of Dextran T-500 (Pharmacia, Piscataway, New Jersey) .
• the medium is prepared as follows: For a final volume of 100L
• Example 5 Comparison of Growth of Drosophila .ACC086. cells in M3+5%FBS. MR-Dl. or MR-Dl. and the production of gpl20 in these media
• Drosophila cells (cell line ACC086) (2xl0 6 cells per milliliter) that had been transfected to express human immunodeficiency virus-1 protein gpl20 were cultured in M3 medium with 5% fetal bovine serum (FBS) ; MR-Dl, the medium of Example 2; or MR-D2, the medium of Example 3.
• M3 medium is the medium described in Example 1 with 1 gram per liter yeastolate (Difco, Detroit, Michigan) added.
• Each of the media also contained 300 ⁇ g/ml hygromycin B (HB) , an antibiotic. Cells were cultured for 14 days and the number of cells and amount of gpl20 were determined at intervals.
• MR-D2 medium cell density was 2.7xl0 7 and the concentration of gpl20 was 2,750 ng/ml. In contrast the density of cells grown in M3+5% FBS medium was 2.1xl0 7 and the concentration of gpl20 was 1,207 ng/ml.
• Drosophila cells (2 x 10 ⁇ cells per milliliter) that had been transfected to express human immunodeficiency virus-1 protein gpl20 (cell line ACC086) were cultured in a 250 ml SP flask containing 120 ml of either MD-R2, the medium of Example 3; or MD- R3, the medium of Example 4. Cells were passaged at 7 day intervals, and were cultured through 41 passages.
• MR-D2 After 25 passages, in MR-D2 medium there were approximately 23 x 10 ⁇ cells. In MR-D3 medium there were approximately 19 x 10 ⁇ cells per milliliter. After 26 passages, gpl20 production was about 550 ng/ml in MR- D2 and 700 ng/ml in MR-D3. After 30 passages there were approximately 20 x 10 ⁇ cells per milliliter in MR-D2 and approximately 21 x 10 6 cells per milliliter in MR-D3. After 33 passages there were approximately 20 x 10 6 cells per milliliter in both MR-D2 and MR-D3 media, and gpl20 production was about 750 ng/ml in MR-D3 and 650 ng/ml in MR-DC.
• g ⁇ l20 production was about 550 ng/ml in MR-D2 medium, and about 500 ng/ml in MR-D3 medium.
• gpl20 production was about 375 ng/ml in MR-D2 medium and about 525 ng/ml in MR-D3 medium.
• gpl20 production was about 300 ng/ml in MR-D2 medium and about 425 ng/ml in MR-D3 medium.

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• 1991
  • 1991-09-20 EP EP91919121A patent/EP0550678B1/de not_active Expired - Lifetime
  • 1991-09-20 ES ES91919121T patent/ES2112866T3/es not_active Expired - Lifetime
  • 1991-09-20 DE DE69128538T patent/DE69128538T2/de not_active Expired - Lifetime
  • 1991-09-20 WO PCT/US1991/006851 patent/WO1992005247A1/en active IP Right Grant
  • 1991-09-20 CA CA002091444A patent/CA2091444C/en not_active Expired - Lifetime
  • 1991-09-20 AU AU88449/91A patent/AU663060B2/en not_active Expired
  • 1991-09-20 JP JP51755491A patent/JP3224539B2/ja not_active Expired - Lifetime
  • 1991-09-20 AT AT91919121T patent/ATE161575T1/de not_active IP Right Cessation
  • 1991-09-20 DK DK91919121.3T patent/DK0550678T3/da active
  • 1991-09-23 NZ NZ239899A patent/NZ239899A/xx not_active IP Right Cessation
  • 1991-09-23 ZA ZA917559A patent/ZA917559B/xx unknown
  • 1991-09-24 IE IE334691A patent/IE913346A1/en not_active IP Right Cessation
  • 1991-09-25 PT PT99047A patent/PT99047B/pt not_active IP Right Cessation
  • 1991-09-25 MX MX9101253A patent/MX9101253A/es unknown
• 1998
  • 1998-03-17 GR GR980400592T patent/GR3026395T3/el unknown
  • 1998-06-29 HK HK98108792A patent/HK1008751A1/xx not_active IP Right Cessation

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